Human CD94/NKG2A is an inhibitory receptor that recognizes HLA-E and is expressed by NK cells and a subset of T cells. To elucidate the cell surface dynamics of CD94/NKG2A receptors, we have expressed CD94/NKG2A-EGFP receptors in the rat basophilic leukemia (RBL) cell line. Photobleaching experiments revealed that CD94/NKG2A-EGFP receptors move freely within the plasma membrane and accumulate at the site of contact with ligand. The enriched CD94/NKG2A-EGFP is markedly less mobile than the nonligated receptor. We observed that not only are lipid rafts not required for receptor polarization, they are excluded from the site of receptor contact with the ligand. Furthermore, the lipid raft patches normally observed at the sites where FcepsilonR1 activation receptors are cross-linked were not observed when CD94/NKG2A was coengaged along with the activation receptor. These results suggest that immobilization of the CD94/NKG2A receptors at ligation sites not only promote sustenance of the inhibitory signal, but by lipid rafts exclusion prevent formation of activation signaling complexes. Also, we have shown that the orphan gene, NKG2F, can be expressed as protein, and that expression appears to be confined to intracellular compartments probably due to its inability to associate with CD94. It can however associate with DAP12 thereby providing activation signaling potential. In terms of gene regulation, we have identified the core promoters of human CD94, which contain conserved GAS/EBS elements that can form protein-DNA complexes. The two promoters differentially regulate expression of the CD94 gene in response to treatment with IL-2 or IL-15. Clarification of the precise roles of these two promoters and the factors that regulate them is under active investigation. Nk cells are notoriously difficult to transfect, a fact that has hampered NK cell biology studies. To overcome this, we developed procedures for the newly available Amaxa nucleofection system that allow for efficient transfection of NK cells.